NetNotes
Edited by Bob Price University of South Carolina School of Medicine
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in the Microscopy (
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Precipitation Observed After Making Freeze Substitution Cocktail Containing 5% Water and Use of Molecular Sieves 3DEM Listserver We recently tried to make a freeze substitution cocktail
consisting of 1% osmium tetroxide, 0.1% uranyl acetate and 5% water, but observed that the mixture turned slightly cloudy within 1–2 minutes. We tried to see if the precipitation would occur in the absence of osmium tetroxide and observed some cloudiness aſter mixing uranyl acetate solution in water with pure acetone. Just wondering if anyone has encountered a simi- lar problem and if so, we would appreciate some tips on how to overcome this problem. Tank you! Qiaohui Yang qiaohui.
yang@ymail.com
Mix the osmium, water and acetone and cool to -20°C.
Ten add the uranyl acetate from a stock, e.g., 20% in metha- nol. Cooling the mixture in my experience stops the uranyl acetate from precipitating. Christopher Peddie christopher.
peddie@crick.ac.uk
At best, the solubility of UA is around 7–8% in water. It’s
best to make a 5% stock solution, dissolved in water, for exam- ple, and add the quantity of 5% UA to the acetone. Te precipi- tation of UA is likely due to the acetone as UA is not soluble in acetone. Kimberley Gibson
kimberley.gibson@
yale.edu
I would agree with the 7–8% stock concentration for UA.
Even at that concentration, it would take a while to dissolve in methanol. Additionally, I would filter it with a 0.22-micron filter, make aliquots and then freeze in LN2 LN2
. I don’t know if
is overkill but I have never seen cloudy precipitation in my freeze substitution mixes and I was using 3% OsO4 and 0.5%
UA along with some tannic acid and glutaraldehyde. Hope this helps! Amar Parvate
aparvate@lji.org
Some people do not mention the solvent in their comments
(or in the Methods and Materials in their papers). In theory, you can push the stock solution in pure water to 7–8%,
70 doi:10.1017/S1551929520000814
but this depends on whether you have CO2 free water. Te CO2
before dissolving the
/carbonate anion is one of our enemies in these solutions. You may boil water to get rid of CO2
UA. Or use freshly distilled (!!) water. Yes, distilled, not the Millipore stuff. You may also add minute amounts (a mini drop
of glacial acetic acid) which reduces the pH such that the CO2 is favored against the bicarbonate anion and the carbonate. Solubility also depends on the temperature. Tus, I do not recommend placing stock solutions into the cold room, below 10°C, or fridge. Nor does it make sense to freeze them. Make small amounts and use them up. Freezing causes precipitation. UA dissolves nicely in MeOH, up to 20%, but this does not last that long. I would make small amounts which are needed within a week Reinhard Rachel
reinhard.rachel@biologie.uni- regensburg.de
Tere are two solutions to your problem. One is to make
the freeze-substitution mixture without water (so for 5 ml end volume dissolve the osmium and uranyl acetate in 4.75 ml ace- tone), cool it to -90°C and then pipette in the water (this needs to be done quickly otherwise the water freezes in the pipette tip). Ten vortex the solution until all visible ice crystals have dissolved and cool the mixture again. Te other solution is to use a stock solution of 20% uranyl acetate dissolved in dry methanol. Tis was also done in the Walther & Ziegler paper on the addition of water in freeze substitution. In my hands, using the first method, 1% water is enough to enhance mem- brane contrast, 3% enhances the visibility of glycogen, and no additional contrast is gained when using 5%. Te small amount of methanol added in method 2 allows for more water to be dis- solved in the substitution mix at lower temperatures, so more water needs to be added to get the same contrast enhancement effect. Rob Mesman
r.mesman@
science.ru.nl
Following up on this topic, I’d like to ask if you are prepar-
ing the dry ethanol and/or acetone yourself, and if so—how do you do it? We are using molecular sieves, which we wash (and store) in alcohol to remove abrasives/dust, then place it into dialysis tubes (with clips to close both ends) and add this to bottles in which the alcohol is supplied. We refill a bottle once. However, the handling of these dialysis-tubes is a pain and they tend to burst when introduced into the bottles. Sev- eral suppliers sell extra-dry ethanol or acetone with molecular sieves floating freely in the bottles. We had a discussion in the lab, whether abrasive dust from molecular sieves is transferred onto the samples, and whether this happens to an extent that influences imaging, e.g., for critically-point dried samples, as
www.microscopy-today.com • 2020 May
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